Project Summary During embryogenesis, organ precursors must be precisely positioned to allow the critical inductive tissue- tissue interactions which drive cell fate specification to occur. How heart field precursors are positioned during development, however, remains poorly understood. The current model assumes that all myocardial and endocardial cells arise from the primary and secondary (or anterior) heart fields (1HF and 2HF). We have recently found however that: (i) many cells of the atrial and atrioventricular junction myocardium, the pacemaker, inflow, and proepicardium do not originate in either HF; and (ii) surprisingly, they are derived from the lateral plate mesoderm (designated as the tertiary HF (3HF) hereafter) posterior to the 1HF and 2HF. Importantly, our preliminary study has revealed that: (iii) precursors of the 3HF as well as the 1HF and 2HF are among the first cells to form the primitive streak; (iv) they exit from the primitive streak bilaterally without crossing the embryonic midline and (v) migrate with little lateral cell intercalation until their arrival at each HF. Thus, an original position of HF precursor cells withn the PS predicts their defined destination within the left or right. Despite the importance of PS-origin for all three HFs, little is known about how the HF precursors populate to the defined position within the PS and how they migrate symmetrically to form the three HFs bilaterally. Further, our previous studies have identified that: (vi) the precursors of the 1HF and 2HF reside at the posterior end of the blastodisc; (g) individual precursor cells undergo an oriented cell division and generate a series of daughter cells that are aligned as an array along the embryonic AP axis of the primitive streak; (vii) midline cells of the primitive streak uniquely undergo cell death: and finally (viii) inhibition of the midline cell death results in randomizatio of heart looping. These findings lead to a novel model for heart field mesodermal patterning whereby oriented cell division in the PS coupled with midline cell death precisely position HF precursors prior to gastrulation. This proposal will test this model by determining: the origin and formation of three HFs from the primitive streak (Aim 1), the ability of a oriented cell division t drive precursors of individual HFs to defined domains within the primitive streak (Aim 2), and the regulatory role of midline cell death for defining the left and right migration pathways of HF precursors to form bilateral HFs (Aim 3). Taken together, the proposed study will provide the first understanding of how cardiac precursors gastrulate in amniotes, while establishing the importance of coordinated positioning in the primitive streak to ultimate cardiogenic fate.